Two general approaches are used for introduction of new heritable genetic information (“transformation”) into cells. One approach is to introduce the new genetic information as part of another DNA molecule, referred to as an “episomal vector,” or “mini-chromosome”, which can be maintained as an independent unit (an episome) apart from the host chromosomal DNA molecule(s). Episomal vectors contain all the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Many episomal vectors are available for use in bacterial cells (for example, see Maniatis et al., “Molecular Cloning: a Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982). However, only a few episomal vectors that function in higher eukaryotic cells have been developed. Higher eukaryotic episomal vectors were primarily based on naturally occurring viruses. In higher plant systems gemini viruses are double-stranded DNA viruses that replicate through a double-stranded intermediate upon which an episomal vector could be based, although the gemini virus is limited to an approximately 800 bp insert. Although an episomal plant vector based on the Cauliflower Mosaic Virus has been developed, its capacity to carry new genetic information also is limited (Brisson et al., Nature, 310:511, 1984.).
The other general method of genetic transformation involves integration of introduced DNA sequences into the recipient cell's chromosomes, permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes. The introduced DNA usually can be broken and joined together in various combinations before it is integrated at random sites into the cell's chromosome (see, for example Wigler et al., Cell, 11:223, 1977). Common problems with this procedure are the rearrangement of introduced DNA sequences and unpredictable levels of expression due to the location of the transgene integration site in the host genome or so called “position effect variegation” (Shingo et al., Mol. Cell. Biol., 6:1787, 1986). Further, unlike episomal DNA, integrated DNA cannot normally be precisely removed. A more refined form of integrative transformation can be achieved by exploiting naturally occurring viruses that integrate into the host's chromosomes as part of their life cycle, such as retroviruses (see Chepko et al., Cell, 37:1053, 1984).
One common genetic transformation method used in higher plants is based on the transfer of bacterial DNA into plant chromosomes that occurs during infection by the phytopathogenic soil bacterium Agrobacterium (see Nester et al., Ann. Rev. Plant Phys., 35:387-413, 1984). By substituting genes of interest for a portion of the naturally transferred bacterial sequences (called T-DNA), investigators have been able to introduce new DNA into plant cells. However, even this more “refined” integrative transformation system is limited in three major ways. First, DNA sequences introduced into plant cells using the Agrobacterium T-DNA system are frequently rearranged (see Jones et al., Mol. Gen. Genet., 207:478, 1987). Second, the expression of the introduced DNA sequences varies between individual transformants (see Jones et al., Embo J., 4:2411-2418, 1985). This variability is presumably caused by rearranged sequences and the influence of surrounding sequences in the plant chromosome (i.e., position effects), as well as methylation of the transgene. Finally, insertion of extra elements into the genome can disrupt the genes, promoters or other genetic elements necessary for normal plant growth and function.
Another widely used technique to genetically transform plants involves the use of microprojectile bombardment to integrate DNA sequences into the genome. In this process, a nucleic acid containing the desired genetic elements to be introduced into the plant's native chromosome is deposited on or in small metallic particles, e.g., tungsten, platinum, or preferably gold, which are then delivered at a high velocity into the plant tissue or plant cells. However, similar problems arise as with Agrobacterium-mediated gene transfer, and as noted above expression of the inserted DNA can be unpredictable and insertion of extra elements into the genome can disrupt and adversely impact plant processes.
One attractive alternative to the commonly used methods of transformation is the use of an artificial chromosome. Artificial chromosomes are episomal nucleic acid molecules that exist autonomously from the native chromosomes of the host genome. They can be linear or circular DNA molecules that are comprised of cis-acting nucleic acid sequence elements that provide replication and partitioning activities (see Murray et al., Nature, 305:189-193, 1983). Desired elements include: (1) origin of replication, which are the sites for initiation of DNA replication, (2) centromeres (site of kinetochore assembly and responsible for proper distribution of replicated chromosomes into daughter cells at mitosis or meiosis), and (3) if the chromosome is linear, telomeres (specialized DNA structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule). An additional desired element is a chromatin organizing sequence. It is well documented that centromere function is crucial for stable chromosomal inheritance in almost all eukaryotic organisms (reviewed in Nicklas 1988). The centromere accomplishes this by attaching, via centromere binding proteins, to the spindle fibers during mitosis and meiosis, thus ensuring proper gene segregation during cell divisions.
Artificial chromosomes have been engineered using one of two approaches. The first approach identifies and assembles the desired chromosomal elements into an artificial construct. This approach has been described as “bottom-up” and involves the use of a heterologous system (i.e. bacteria or fungal) to perform the various cloning steps necessary to assemble the artificial chromosome. Artificial chromosomes of this type will be referred to in this application as “mini-chromosomes”. The second approach derives the artificial chromosome from existing chromosomes through chromosome fragmentation and, optionally, subsequent addition of desired elements including transgenes. For example, an existing chromosome can be induced to undergo breakage events that result in chromosomal fragments. Minimal fragments that possess the elements for replication and segregation during cell division (e.g. centromere, origins of replication and/or telomeres) can be identified. These derived artificial chromosomes can then be used as targets for further manipulation including the addition of one or more transgenes. This approach has been described as “top-down” and does not require the use of a heterologous system (e.g. bacterial or fungal) since it doesn't require in vitro-based cloning steps. Artificial chromosomes of this type will be referred to in this application as “recombinant chromosomes”.
The essential chromosomal elements for construction of artificial chromosomes have been precisely characterized in lower eukaryotic species, and more recently in mouse and human. Autonomous replication sequences (ARSs) have been isolated from unicellular fungi, including Saccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe (see Stinchcomb et al., 1979 and Hsiao et al., 1979). An ARS behaves like an origin of replication allowing DNA molecules that contain the ARS to be replicated in concert with the rest of the genome after introduction into the cell nuclei of these fungi. DNA molecules containing these sequences replicate, but in the absence of a centromere they are not partitioned into daughter cells in a controlled fashion that ensures efficient chromosome inheritance.
Artificial chromosomes have been constructed in yeast using the three cloned essential chromosomal elements (see Murray et al., Nature, 305:189-193, 1983). None of the essential components identified in unicellular organisms, however, function in higher eukaryotic systems. For example, a yeast centromere sequence will not confer stable inheritance upon vectors transformed into higher eukaryotes.
In contrast to the detailed studies done in yeast, less is known about the molecular structure of functional centromeric DNA of higher eukaryotes. Ultrastructural studies indicate that higher eukaryotic kinetochores, which are specialized complexes of proteins that form on the centromere during late prophase, are large structures (mammalian kinetochore plates are approximately 0.3 μm in diameter) which possess multiple microtubule attachment sites (reviewed in Rieder, 1982). It is therefore possible that the centromeric DNA regions of these organisms will be correspondingly large, although the minimal amount of DNA necessary for centromere function may be much smaller.
While the above studies have been useful in elucidating the structure and function of centromeres, it was not known whether information derived from lower eukaryotic or mammalian higher eukaryotic organisms would be applicable to Sugarcane. There exists a need for cloned centromeres from Sugarcane, which would represent a first step in the production of Sugarcane artificial chromosomes, or in the identification of Sugarcane recombinant chromosomes. There further exists a need for Sugarcane cells, plants, seeds and progeny containing functional, stable, and autonomous artificial or recombinant chromosomes capable of carrying a large number of different genes and genetic elements.